Objective:To investigate the repair effect of bone marrow mesenchymal stem cells(BMSCs)on interferon gamma(IFN-γ)induced ovarian granulosa cell immune injury.Methods:A model of IFN-γ-induced ovarian granulosa cell immune injury was estab-lished.KGN cells after modeling were co-cultured with human BMSCs(hBMSCs)and divided into three groups:negative control(NC)group,IFN-γ group,and BMSC group.After co-culture,cell proliferation was determined by a cell counting kit 8 assay for 3 consecu-tive days.Cell apoptosis was determined using an Annexin V-FITC apoptosis detection kit and a CytoFLEX flow cytometer.The estra-diol level and the mRNA expression levels of aromatase CYP19A1 and FSHR were measured to evaluate the hormone synthesis ability.The level of lactate dehydrogenase(LDH),mRNA expression levels of NLRP3,caspase-1(CASP1),and interleukin-1β,and protein expression levels of NLRP3,CASP1,and gasdermin-D were determined to evaluate cell pyroptosis.Results:The proliferation rate of granulosa cells in the IFN-γ group and the BMSC group was lower than that in the NC group at 24 h,48 h and 72 h,and that in the IFN-γ group was lower than that in the BMSC group(P<0.001).The number of apoptotic granulosa cells in the INF-γ group and the BMSC group was higher than that in the NC group,and that in the INF-γ group was higher than that in the BMSC group(P<0.001).The estradiol level in the INF-γ group and the BMSC group was lower than that in the NC group,and that in the INF-γ group was lower than that in the BMSC group(P<0.001).Compared with the IFN-γ group,the mRNA expression of CYP19A1 and FSHR in the BMSC group decreased(P<0.001).Compared with the IFN-γ group,the BMSC group had significantly decreased LDH level,mRNA expression levels of NLRP3 and CASP1,and protein expres-sion level of CASP1.Conclusion:hBMSCs repair the immune in-jury of KGN cells induced by IFN-γ by restoring cell proliferation,inhibiting cell apoptosis,repairing endocrine function,and reducing pyroptosis.

BACKGROUND/OBJECTIVES: Activating brown adipose tissue (BAT) and browning of white adipose tissue (WAT) can protect against obesity and obesity-related metabolic conditions.Cryptotanshinone (CT) regulates lipid metabolism and significantly ameliorates insulin resistance. Adenosine-5'-monophosphate (AMP)-activated protein kinase (AMPK), a receptor for cellular energy metabolism, is believed to regulate brown fat activity in humans.MATERIALS/METHODS: The in vivo study included high-fat-fed obese mice administered orally 200/400 mg/kg/d CT. They were evaluated through weight measurement, the intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test (IPITT), cold stimulation test, serum lipid (total cholesterol, triglycerides, and low-density lipoprotein) measurement, hematoxylin and eosin staining, and immunohistochemistry.Furthermore, the in vitro study investigated primary adipose mesenchymal stem cells (MSCs) with incubation of CT and AMPK agonists (acadesine)/inhibitor (Compound C).Cells were evaluated using Oil Red O staining, Alizarin red staining, flow cytometry, and immunofluorescence staining to identify and observe the osteogenic versus adipogenic differentiation. Quantitative real-time polymerase chain reaction and the Western blot were used to observe related gene expression. RESULTS: In the diet-induced obesity mouse model mice CT suppressed body weight, food intake, glucose levels in the IPGTT and IPTT, serum lipids, the volume of adipose tissue, and increased thermogenesis, uncoupling protein 1, and the AMPK pathway expression. In the in vitro study, CT prevented the formation of lipid droplets from MSCs while activating brown genes and the AMPK pathway. AMPK activator enhanced CT’s effects, while the AMPK inhibitor reversed the effects of CT. CONCLUSION CT promotes adipose tissue browning to increase body thermogenesis and reduce obesity by activating the AMPK pathway. This study provides an experimental foundation for the use of CT in obesity treatment.

To explore the possibility of periodic treatment of high dose asteroidal valerate (E2V) after hysteroscopy surgery on serious intrauterine adhesion. Methods The post-operative effects of periodic high-dose E2V treatment was compared with retrospective analysis on 62 cases of serious intrauterine adhesion and their clinical files. The cases were divided to 3 groups: immediate high-dose periodic treatment of E2V after hysteroscopy electric resection + contraceptive ring fitting (experiment group I), low-dose periodic treatment of E2V (experiment group II), and Irregular use of hormone treatment (control group). Results The effective rate of menstruation recovery of experiment group I and that of experiment group II are higher than control group (P＜0. 05);experiment group I is higher than experiment group II (P＜0. 05). The effective rate of Intrauterine recovery of experiment group I is higher than experiment group II and control group(P＜0. 05); the curative effect of experiment group I is higher than experiment group II (P＜0. 05). The endometrium of experiment group I is thicker than experiment group II and control group (P＜0. 05). Conclusion The use of high dose asteroidal valerate after hysteroscopy surgery on serious intrauterine adhesion, which improves the treatment effectively, is reliable and safe.

Objective To investigate feasibility of CM-Dil labeling mesenchymal stem cells in vitro.Methods We separate,culture and identify rat bone marrow mesenchymal stem cells,after labeling MSCs with CM-Dil concentration respectively in 1,2,4μg/mL,observe label rate of MSCs after 15 min,24 h,48 h,72 h,5 passage and 8 passage,then detect cell cycle with FCM and cell proliferation capability with CCK8.Results MSC cultured with the whole bone marrow adherent method were strongly positive for CD44,CD29 and negative for CD34,CD45.The label rates of MSC 15 min after labeled with CM-Dil 1,2,4 μg/mL were respectively (31.60±1.25)％,(88.73±1.79)％,(96.89±1.31)％,and there has statistically difference (F=1 757.21,P=0.000);the growth curve of the 4 groups was similar,and the percentages of G1 phase,S phase and G2/M phase between the 4 groups have no statistically difference (P＞0.05).Conclusion CM-Dil in concentration 4 μg/mL has high label rate and low cytotoxicity,therefore could be a efficient and stable method of labeling MSCs.

BACKGROUND:Chemotherapy drugs can damage the ovarian function in women of childbearing age, and even lead to premature ovarian failure. Therefore, to improve and restore the ovarian function in patients has become an important issue. OBJECTIVE:To explore the therapeutic effect and feasibility of bone marrow mesenchymal stem cel therapy against chemotherapy-induced ovarian damage. METHODS:Rat models of chemotherapy-induced premature ovarian failure were established, and injected with PKH26-labeled bone marrow mesenchymal stem cels. At 15, 30, 45, 60 days after cel transplantation, five rats were selected respectively to detect folicle-stimulating hormone and estradiol levels, and then, the rats were kiled to take the right ovary for pathological examination. The number of ovarian folicles was detected under light microscope. At 30 days after cel transplantation, another two rats were selected to mate with male rats to observe the difference in the reproductive activity. RESULTS AND CONCLUSION:Four of 22 rats (18%) gradualy recovered their estrous cycle after cel transplantation, with the decreased folicle-stimulating hormone level and increased estradiol level. Moreover, the number of folicles was reduced. Al of these indicated that the ability to have children in rats was not damaged.These experimental findings suggest that bone marrow mesenchymal stem cels can partialy improve the ovarian function of rats under chemotherapy.

OBJECTIVETo investigate the histomorphology and the expressions of the proliferation marker Ki-67 and estrogen receptor in the uterus of mice with autoimmune premature ovarian failure (POF) induced by zona pellucida 3 peptide (pZP3).

METHODSAutoimmune POP models were established in 20 female BALB/c mice (7-8 weeks old) by immunization with pZP3 and another 20 mice served as the control group. The POP models were verified by vaginal cytology, serum sex hormones, ovary histomorphology and ZP3 antibody immunohistochemistry. The histomorphology and expressions of Ki-67, estrogen receptor α and estrogen receptor β in the uterus of the mice were detected.

RESULTSAutoimmune POP models were established successfully in 80% of the mice at 8 weeks after the immunization. Compared with those in the control group, the mice in the model group showed a smaller volume of the uterus, thinner endometrium and a reduced number of glands. The luminal epithelial cells, glandular epithelial cells and stromal cells in the uterus of the model mice all presented with a lower expression of Ki-67 than those in the control group, and Ki-67 translocation from the nuclei to the cytoplasm was found in the model group. The luminal epithelial cells, glandular epithelial cells and stromal cells showed positive ERα immunoreactivity in the model group but not in the control group. No obvious ERβ expression was found in the uterus in either of the groups.

CONCLUSIONpZP3 can induce autoimmune POP, cause suppressed proliferation of the endometrial epithelial cells and stromal cells, and reduce the cellular expression of ERα in the uterus of mice.

Animals ; Autoimmune Diseases ; metabolism ; Cell Nucleus ; Egg Proteins ; Endometrium ; Epithelial Cells ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Female ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Membrane Glycoproteins ; Mice ; Mice, Inbred BALB C ; Primary Ovarian Insufficiency ; metabolism ; Receptors, Cell Surface ; Stromal Cells ; Uterus ; metabolism ; Zona Pellucida Glycoproteins

Objective To investigate the histomorphology and the expressions of the proliferation marker Ki-67 and estrogen receptor in the uterus of mice with autoimmune premature ovarian failure (POF) induced by zona pellucida 3 peptide (pZP3). Methods Autoimmune POP models were established in 20 female BALB/c mice (7-8 weeks old) by immunization with pZP3 and another 20 mice served as the control group. The POP models were verified by vaginal cytology, serum sex hormones, ovary histomorphology and ZP3 antibody immunohistochemistry. The histomorphology and expressions of Ki-67, estrogen receptorαand estrogen receptorβin the uterus of the mice were detected. Results Autoimmune POP models were estblished successfully in 80%of the mice at 8 weeks after the immunization. Compared with those in the control group, the mice in the model group showed a smaller volume of the uterus, thinner endometrium and a reduced number of glands. The luminal epithelial cells, glandular epithelial cells and stromal cells in the uterus of the model mice all presented with a lower expression of Ki-67 than those in the control group, and Ki-67 translocation from the nuclei to the cytoplasm was found in the model group. The luminal epithelial cells, glandular epithelial cells and stromal cells showed positive ERαimmunoreactivity in the model group but not in the control group. No obvious ERβexpression was found in the uterus in either of the groups. Conclusion pZP3 can induce autoimmune POP, cause suppressed proliferation of the endometrial epithelial cells and stromal cells, and reduce the cellular expression of ERαin the uterus of mice.

Objective To investigate the histomorphology and the expressions of the proliferation marker Ki-67 and estrogen receptor in the uterus of mice with autoimmune premature ovarian failure (POF) induced by zona pellucida 3 peptide (pZP3). Methods Autoimmune POP models were established in 20 female BALB/c mice (7-8 weeks old) by immunization with pZP3 and another 20 mice served as the control group. The POP models were verified by vaginal cytology, serum sex hormones, ovary histomorphology and ZP3 antibody immunohistochemistry. The histomorphology and expressions of Ki-67, estrogen receptorαand estrogen receptorβin the uterus of the mice were detected. Results Autoimmune POP models were estblished successfully in 80%of the mice at 8 weeks after the immunization. Compared with those in the control group, the mice in the model group showed a smaller volume of the uterus, thinner endometrium and a reduced number of glands. The luminal epithelial cells, glandular epithelial cells and stromal cells in the uterus of the model mice all presented with a lower expression of Ki-67 than those in the control group, and Ki-67 translocation from the nuclei to the cytoplasm was found in the model group. The luminal epithelial cells, glandular epithelial cells and stromal cells showed positive ERαimmunoreactivity in the model group but not in the control group. No obvious ERβexpression was found in the uterus in either of the groups. Conclusion pZP3 can induce autoimmune POP, cause suppressed proliferation of the endometrial epithelial cells and stromal cells, and reduce the cellular expression of ERαin the uterus of mice.

OBJECTIVETo investigate the effect of lentivirus-mediated Bcl-2 gene transfection in protecting human primary ovarian granulosa cells against phosphoramide mustard (PM)-induced apoptosis.

METHODSGranulosa cells were isolated from the follicle fluid of women undergoing in vitro fertilization and embryo transfer. The lentiviral vectors carrying Bcl-2 gene (pGC-FU-Bcl-2) and enhanced green fluorescence protein (pGC-FU-EGFP) were constructed and packaged into high-titer lentiviruses. The resulting recombinant lentivirus carrying Bcl-2 and EGFP genes or the empty vector were used to infect the primary human ovarian granulosa cells, followed by addition of PM in the cell culture, with untreated granulosa cells as the control. The cell apoptosis was detected by Annexin V and Hochst 33258 staining, and the expression of Bcl-2 protein was assessed using Western blotting.

RESULTSThe control granulosa cells showed an apoptotic rate of (1.93±0.28)%. The cells infected with pGC-FU-Bcl-2 prior to PM exposure had a apoptotic rate of (6.99±10.55)%, significantly higher than that of the control cells, but significantly lower than that of the cells with PM exposure only and those infected with the empty vector before PM exposure (P<0.05). The expression of Bcl-2 was the highest in the cells infected with pGC-FU-Bcl-2 prior to PM exposure (P<0.05).

CONCLUSIONLentivirus-mediated Bcl-2 gene transfection can protect human ovarian granulosa cells against PM-induced apoptosis by upregulating Bcl-2 protein expression.

Adult ; Apoptosis ; drug effects ; Female ; Genes, bcl-2 ; Genetic Vectors ; Granulosa Cells ; drug effects ; Humans ; Lentivirus ; genetics ; Phosphoramide Mustards ; Transfection

Objective To explore the effects of sensory system obstacle on balance ability of patients with hemiplegia after stroke. Methods20 patients with hemiplegia after stroke (experimental group) and 20 normal people (control group) received Sensory Organization Test(SOT) with Smart Equitest Balance Master system. The balance index and the sensory index were accessed. Results The scores of the balanceindex in the experimental group were lower than in the control group in EC, SV, EOSS, ECSS, SVSS (P<0.05). The scores of somatosensory,visual and vestibular indexes in the experimental group were lower than in the control group (P<0.05), especially for vestibular index.Conclusion The sensory loss caused the balance dysfunction in patients with stroke.